Abstract

Vascular smooth muscle cells (VSMCs) are one of the main cellular determinants in arterial pathology. A large body of evidence indicates that death of VSMCs is associated with features of high-risk/vulnerable atherosclerotic plaques. Mitochondrial turnover is an essential aspect of the mitochondrial quality control in which dysfunctional mitochondria are selectively eliminated through autophagy and replaced through expansion of preexisting mitochondria. Even though successful autophagy promotes VSMC survival, it is unclear whether reduced autophagic flux affects mitochondrial quality control of VSMCs in atherosclerotic plaques. By using apolipoprotein E-deficient (ApoE-/-) mice carrying a VSMC-specific deletion of the essential autophagy gene Atg7, we show in the present study that impaired VSMC autophagy promotes an unstable plaque phenotype, as well as the accumulation of fragmented mitochondria with reduced bioenergetic efficiency and more oxidative stress. Furthermore, we demonstrate that disrupted autophagic flux is linked to defective mitophagy and biogenesis of mitochondria, which exacerbate VSMC apoptosis and in turn plaque vulnerability. Overall, our data indicate that mitochondrial quality control is a promising therapeutic target to stabilize atherosclerotic plaques.

a Representative images of consecutive aortic sinus sections immunostained with PINK1, Parkin, P62 (red), α-SMA (green) antibodies, and DAPI (blue, nucleus) from Atg7+/+Tagln-Cre+, ApoE−/− and Atg7F/FTagln-Cre+, ApoE−/− mice after 10 weeks of high-fat diet (HFD). The graph represents the % of PINK1, Parkin, or P62 staining in VSMCs within the plaque area and the data are the mean ± SEM from n = 8 mice/group. ***P < 0.001; Student’s t-test. Scale bar, 20 µm. b Western blot analyses of the expression of PINK1 and Parkin proteins in aortic VSMCs isolated from Atg7+/+Tagln-Cre+, ApoE−/− and Atg7F/FTagln-Cre+, ApoE−/− mice after 10 weeks of HFD, β-actin was used as the loading control. Bands are shown in duplicate. The graph represents the densitometric analysis of the expression level of PINK1 and Parkin proteins. The data are the mean ± SEM of three independent experiments from different primary VSMC cultures per group. **P < 0.01; *P < 0.05; Student’s t-test

a Flow cytometry analyses of mitophagy flux in aortic VSMCs isolated from Atg7+/+Tagln-Cre+, ApoE−/− and Atg7F/FTagln-Cre+, ApoE−/−mice after 10 weeks of high-fat diet (HFD). Cells were incubated either with or without oxidized LDL (200 μg ApoB/mL, left graph) or CCCP (20 µM, right graph) for 8 h and treated either with or without the lysosomal inhibitor Bafilomycin A1 (BafA1) (100 nM). VSMCs were then stained with MitoTR for flow cytometry analysis. The data are expressed as mean ± SEM of five independent experiments (left graph); *P < 0.05; Wilcoxon signed-rank test, ns nonsignificant; and as mean ± SEM of three independent experiments (right graph) *P < 0.05; Mann–Whitney non-parametric test. b Western blot analyses of the expression of TOMM 40 and VDAC1 proteins in aortic VSMCs isolated from Atg7+/+Tagln-Cre+, ApoE−/− and Atg7F/FTagln-Cre+, ApoE−/− mice after 10 weeks of HFD. Cells were incubated either with or without oxidized LDL (200 μg ApoB/mL) for 16 h and treated either with or without the lysosomal inhibitor Bafilomycin A1 (BafA1) (100 nM). β-Actin was used as the loading control. The graph represents the densitometric analysis of the expression level of TOMM 40 and VDAC1 proteins. The data are expressed as mean ± SEM of four independent experiments from different primary VSMC cultures. *P < 0.05; ***P < 0.01; one-way ANOVA, Bonferroni’s multiple comparison test. c Western blot analyses of the expression of TFEB and PGC-1-α proteins in aortic VSMCs isolated from Atg7+/+Tagln-Cre+, ApoE−/− and Atg7F/FTagln-Cre+, ApoE−/− mice after 10 weeks of HFD. Cells were incubated either with or without oxidized LDL (200 μg ApoB/mL) for 8 h. β-Actin was used as the loading control. The graph represents the densitometric analysis of the expression level of TFEB and PGC-1-α proteins. The data are expressed as mean ± SEM of four independent experiments from different primary VSMC cultures. *P < 0.05; ***P < 0.001; Student’s t-test. (d) Representative images of TFEB (green) and DAPI (blue, nucleus) immunostaining in aortic VSMCs isolated from Atg7+/+Tagln-Cre+, ApoE−/− and Atg7F/FTagln-Cre+, ApoE−/− mice after 10 weeks of HFD. Cells were incubated either with or without oxidized LDL (200 μg ApoB/mL) for 8 h. The graph represents the intensity of nuclear TFEB fluorescence and the data are the mean ± SEM of three independent experiments from different primary VSMC cultures. *P < 0.05; ###P < 0.001; one-way ANOVA, Kruskal–Wallis non-parametric test. e Representative images of cleaved-caspase 3 (green) and DAPI (blue, nucleus) immunostaining in aortic VSMCs isolated from Atg7+/+Tagln-Cre+, ApoE−/− and Atg7F/FTagln-Cre+, ApoE−/− mice after 10 weeks of HFD. Cells were incubated with or without oxidized LDL (200 μg ApoB/mL) for 16 h. Scale bar, 20 µm. The graph represents the % of cleaved-caspase 3-positive cells and the data are the mean ± SEM of three independent experiments from different primary VSMC cultures. *P < 0.05; two-way ANOVA, Bonferroni’s multiple comparison test. Scale bar, 20 µm